Highly pathogenic avian (HPAI) H5N1 influenza viruses are associated with a 50% mortality rate in humans and are considered a primary pandemic threat. Although the mechanism by which these viruses cause severe disease is not fully defined, an overly aggressive immune response is a hallmark of H5N1 disease. Macrophages, though critical for protection against influenza virus infection, have been implicated in severe H5N1 disease primarily through the production of excessive levels of pro-inflammatory cytokines. The preliminary evidence for this proposed project demonstrates that a defining feature of HPAI H5N1 influenza viruses is their ability to productively replicate in macrophages in a hemagglutinin (HA)-dependent manner while non-H5 influenza viruses are degraded in the cytoplasm upon viral entry. Further, the ability of H5N1 viruses to replicate in macrophages is associated with disease severity in vivo. It is unclear how H5N1 influenza viruses subvert cellular pathways which restrict influenza virus replication in macrophages. This proposal tests the hypothesis that the cellular pathway of autophagy restricts non-H5 influenza virus replication in macrophages by targeting them for degradation in the lysosome while H5N1 viruses fail to activate autophagy, thus being enabled to complete the viral replication cycle. Further, we hypothesize that H5N1 influenza virus replication in macrophages alters cellular functions. The hypothesis will be tested using a parental H1 influenza virus and a reverse genetics-derived virus expressing an H5 HA protein that has been deselected to permit its safe use under BSL2 laboratory conditions. Specific Aim 1 tests the model that autophagy restricts non-H5 influenza virus replication. A combination of confocal imaging and western blots will be used to detect autophagy activation during virus infection. Infections will be carried out in the presence of autophagy inhibitors and virus replication will be determined by quantitative real-time PCR. Specific Aim 2 tests the hypothesis that macrophage function is impaired by replication of H5N1 viruses. Proinflammatory cytokine and nitric oxide levels will be determined by enzyme-linked immunosorbent assay (ELISA) and a phagocytosis assay will be used to determine changes in macrophage function in cells infected with viruses that do or do not replicate in that cell type. The data generated by this proposal will not only increase our understanding of how H5N1 influenza viruses cause excessive disease, but will also increase our understanding of how microbes hijack antiviral pathways in macrophages to support their own replication. Thus, the proposed project will potentially have an important impact on the broader field of pathogens that infect macrophages.